The protein PPAR (peroxisome proliferator activated receptor) is known as a nuclear receptor. Peroxisome which is an intracellular organelle is involved in oxidation and present in the liver and kidneys at large amounts. Also it oxidizes an intracellular fatty acid to produce peroxides which serve to neutralize toxic substances. In addition, it has a function of decomposing excess hydrogen peroxide into water and oxygen by catalase, an oxidase. A peroxisome proliferator signifies a compound capable of increasing the number of peroxisomes, and includes fat and fatty acid, and fibrate and prostaglandin as hyperlipemia-treating agents.
Nuclear receptors having such peroxisome proliferators as ligands are collectively called PPARs, which are divided into PPAR alpha, PPAR delta/beta and PPAR gamma. As known in the art, PPAR alpha is mainly expressed in the liver, and involved in the oxidation of fatty acids or the neutralization of toxic substances, and related to inflammatory reaction. PPAR delta/beta is found to distribute uniformly and to be involved in embryonic development, and PPAR gamma is known to be involved in the differentiation and accumulation of fat cells.
It was recently found that the expression of PPAR gamma was increased in a differentiation process of a fat cell line into a fat cell, and that expression of PPAR gamma in fibroblast having no differentiation ability resulted in differentiation of the fibroblast into a fat cell. Particularly, PPAR gamma2 is divided into two isoforms and reported to be specifically expressed only in fat cells at large amounts. mRNA of mouse PPAR gamma1 is coded by eight exons, whereas mRNA of PPAR gamma2 is coded by seven exons. The 5′ untranslated sequence of mRNA of mouse PPAR gamma1 is coded by two exons, whereas the 5′ untranslated sequence of PPAR gamma2 and the additional N-terminal amino acids are coded by one exon. The two isoforms are yielded by alternative promoter use and different splicing, and increase the variety of ligands and make tissue-specific expression possible (Zhu Y et al., 1995).
Meanwhile, type 2 diabetes is characterized by the insulin resistance of skeletal muscle tissues, liver tissues and fat tissues, etc. In the early 1980's, among treating agents of type 2 diabetes, glitazones and the like belonging to thiazolidinediones (TZD) were first reported as a drug which allows glucose level to be reduced and insulin resistance to be improved without stimulating insulin secretion in an experimental rat model of type 2 diabetes.
As glitazones, treating agents of type 2 diabetes, are found to be PPRA gamma agonists (Lehmann J M et al, 1995), it is reported that PPRA gamma ligands (agonists) can improve insulin resistance.
This fact seems to prove the therapeutic effect of the PPAR gamma agonists and to suggest the utility of a system for screening diabetes-effective substances by ELISA, which was established by the present inventors.
Meanwhile, in order to examine how the activity of PPAR gamma in fat cells influences the metabolism of glucose in the muscles and liver, the mechanisms of PPAR gamma agonists as agents for improving diabetes will now be described. First, PPAR gamma protein in fat cells is known as regulating the release of endocrine signal molecules influencing the metabolism of glucose in the muscles and liver, such as cytokine TNF-α or leptin. Expression of the two signal molecules is inhibited by the PPAR gamma agonists in fat cells, and it was found that TNF-α resulted in the increase of insulin resistance and leptin interfered-with insulin signal transmission in any cells (Cohen B. et al., 1996; and Muller G. et al., 1997). This seems to increase insulin resistance. Thus, insulin resistance caused by such two signal molecules can be improved by the PPAR gamma agonists.
Second, there is the antihyperglycemic effect of PPAR gamma agonists. Generally, glucose and fatty acids compete with each other for an energy substrate in muscles, so that the increase of the amount of fatty acids results in the decrease of glucose consumption. Thus, it is believed that the increase of free fatty acids and the increase of glucose synthesis or gluconeogenesis are connected with each other. In this case, the PPAR gamma agonists stimulate fat cells to absorb and store fatty acids, thereby reducing the amount of circulating triglycerides and free fatty acids. Furthermore, the PPAR gamma agonists are known as having an indirect effect on glucose metabolism, reducing the level of fatty acids in the muscles or liver (Martin G. et al., 1998). Accordingly, the PPAR gamma agonists stimulate fatty acid flow from the muscles or liver to white fatty acids and show a dramatic effect on energy expenditure, thereby causing the reduction of gluconeogenesis in the liver and the increase of glucose consumption in muscles.
Finally, PPAR gamma is also expressed in the muscles and liver at a lower expression level than in fat cells and can show an effect of improving diabetes by its direct activation. Namely, it is reported that treatment of experimental rats deficient in fat tissues with troglitazone as a PPAR gamma agonist reduces hyperglycemia and increases insulin sensitivity. This mechanism can be regarded as the role of PPAR gamma agonists generated by a pathway separate from fat cells (Burant C F et al., 1997).
And the PPAR gamma agonists show a direct or indirect effect in various tissues including the muscles and liver.
Moreover, for atheromatous lesions, PPAR gamma is known as being expressed in macrophages including foam cells at a high level.
The foam cells generally means cholesterol-laden cells converted from macrophages embedded in the inner arterial wall. This conversion of the macrophages into the foam cells is regarded as a definite symptom of occurrence of arteriosclerosis.
The conversion process of the cells is known as having a connection with the internalization of oxLDL particles by scavenger receptors, such as CD36 (cell adhesion molecule) and scavenger receptor-A. It was recently reported that PPAR gamma was also closely connected with this process (Tontonoz P. et al., 1998).
For example, it is known that treatment of a human acute monocytic leukemia cell line (THP-1) with PPAR gamma and RXR alpha (retinoid X receptor alpha) agonists induces the expression of PPAR gamma2 and CD36 and promotes the absorption of oxLDL, and treatment of the aorta of experimental rats with the PPAR gamma agonist increases the expression of CD36. Particularly, 9-HODE and 13-HODE, two components of oxLDL, were found to be PPAR gamma agonists. Accordingly, PPAR gamma is an important component of oxLDL-PPAR gamma-CD36 contributing to the accumulation of lipids induced from oxLDL by macrophages. These results indicate that PPAR gamma agonists have the possibility of promoting the formation of foam cells, but clinical data show that glitazone protects patients with type 2 diabetes from arteriosclerosis which can frequently secondarily occur in these patients. This is because treatment of low density lipoprotein (LDL) receptor-deficient rats (arteriosclerosis model) with rosiglitazone and GW784 is known as inhibiting the formation of atheromatous lesion in spite of the increase of CD36 expression.
According to recent studies, it was found that the release of cholesterol from macrophages was controlled by ABCA1 (ATP binding cassette A1), a member of ATP binging cassettes (ABC) of energy-dependent transporter proteins, in which ABCA1 is mutated in patients with tangier disease caused by cholesterol accumulation in macrophages and other reticuloendothelial cells. The transcription of an ABCA1 gene is regulated by a nuclear oxysterol receptor LXR (liver X receptor), and PPAR gamma and LXR agonists act together to induce the expression of ABCA1 and to promote the release of cholesterol from macrophages and THP-1 cell lines induced from rat embryonic stem cells (Chawla A. et al., 2001).
Furthermore, the ABCA1 gene is known as a direct target gene of a PPAR gamma/RXR heterodimer and causes the expression of LXR-alpha when the activity of PPAR gamma is increased. This leads to the increase of ABCA1 expression and the release of cholesterol. In addition, PPAR gamma is known to regulate the introduction and release of cholesterol esters in macrophages. By this effect, oxLDL will be removed which increases the release of free cholesterol introduced to the liver, and causes aortic lesions by absorption into macrophages. Particularly, the PPAR gamma agonists seem to interfere with the development arteriosclerosis in vivo by producing HDL (high density lipoprotein) in the human and increasing the release of cholesterol from macrophages and endothelial cells.
Moreover, there is the broad role of PPAR gamma in the regulation of inflammatory reaction of monocytes/macrophages.
Treatment of PPAR gamma including 15d-PGJ2 and glitazone, with monocytes or macrophages, reduces the expression of pro-inflammatory cytokine such as TNF-alpha and IL-6 and inhibits the activity of macrophages. However, the PPAR gamma agonists induce an effect of inhibiting inflammatory reaction at a different concentration from a concentration required to activate PPAR gamma in cell-based assays. In addition, any potential PPAR gamma agonists have no effects, but 15d-PGJ2 is the most powerful inhibitor against the cytokine production of monocytes or macrophages in vitro. This suggests that the anti-inflammatory effect of the PPAR gamma agonists can be mediated by PPAR-independent mechanism. This hypothesis is supported by several recent studies. Namely, it was found that glitazone inhibited cytokine production in rats treated with LPS (lipopolysaccharide) and that several different PPAR gamma agonists (15D-PGJ2 and rosiglitazone) inhibited cytokine production even in embryonic stem cells having PPAR gamma+/+ or PPAR gamma± or PPAR gamma−/−, as in macrophages.
Meanwhile, 15d-PGJ2 was recently found to inhibit NF-κB activity (Rossi A. et al., 2000; and Straus D. S. et al., 2000). NF-κB causes acute inflammatory reaction by the covalent modification of its DNA-binding domain and IκB kinase as its regulatory subunit. This suggests that PPAR gamma is not substantially effective against acute inflammations caused by leucocytes.
Furthermore, hypertension is one of metabolic defects which often accompany obesity and type 2 diabetes, etc. Its pathogenesis is complex and connected with blood pressure dysregulation, insulin sensitivity, vascular function, and lipid metabolism.
Recent genetic analysis shows that a PPAR gamma dominant negative mutant is connected with severe hypertension Treatment of an animal model of hypertension with PPAR gamma agonists shows low blood pressure, but it is not yet known that the PPAR gamma agonists are involved in a mechanism forming the basis of an anti-hypertensive effect. However, the fact glitazone reduces blood pressure in the human with no diabetes and an animal model of hypertension having no connection with insulin resistance indicates that the anti-hypertensive effect of PPAR gamma agonists is independent of insulin-sensitizing actions.
Since PPAR gamma is expressed in intra-vascular endothelial cells, the PPAR gamma agonists are considered as improving hypertension by regulating the expression of vascular factors connected with the maintenance of vascular tone, such as type C nutriuretic peptide, endothelin, and plasminogen activator inhibitor-1 (Itoh H. et al., 1999).
Furthermore, the proliferation inhibition and pro-differentiation effects of the PPAR gamma agonists suggest that these compounds can be used as an agent for inhibiting the proliferation of de-differentiated tumor cells. This hypothesis supports the experimental results showing that transplantation of BNX triple immuno-deficient nude mice with breast cancer cells (Elstner E. et al., 1998) and prostate tumor cells (Kubota T. et al., 1998) followed by treatment with TZDs inhibits the proliferation of such tumor cells.
These effects show that a differentiation program according to the result of PPAR-mediated activation can be used even in nonadipogenic lineage colonic cells and inhibits the development of cancer. In contrast, when a transformed experimental mouse deficient in one copy of a gene coding for an APC (adenomatous polyposis coli tumor suppressor is treated with PPAR gamma agonists of a significantly higher amount than one required to increase insulin activity, the PPAR gamma agonists promote the development of colonic tumor (Lefebvre A. M. et al. 1998). A human colon cancer cell line studied by Sarraf and his research team has both normal and malfunctioning APCs, and it is considered that many unknown factors required for colonic cell proliferation in an experimental rat model are involved therein.
Furthermore, a recent study conducted by Pilot showed that administering the PPAR gamma agonists to patients with solid liposarcoma caused antineoplastic pro-differentiation. These agonists reduce the proliferation rate of cancer cells, and thus, it is expected that they will make the progression of this disease slow. In any human colon cancers, it was observed that functions of PPAR gamma were lost due to the PPAR gamma mutation (Sarrf P. et al., 1999). The PPAR gamma agonists induce growth arrest and also the synthesis of different markers of human colon cancer cells in cell culture. This discovery suggests that PPAR gamma inhibits cellular transformation.
Moreover, it is found that the PPAR agonists can be used as inhibitors against angiogenesis, a process necessary for solid-tumor growth, and a metastasis process. Thus, such evidences show that PPAR gamma activation inhibits the growth and development of cancer, and PPAR gamma ligands or agonists will provide a new aim for therapeutic application.
Meanwhile, immunoassays are used for detecting such substances.
Generally, immunoassays using antigen-antibody reaction are known as methods of qualitatively and quantitatively analyzing a biological substance to be measured, in which a specific antibody to an antigen substance to be measured is made such that it can be bound to the antigen, and the binding of the antibody to the antigen is measured with various labels which can recognize and measure the antigen-antibody complex with a device.
Immunoassays of biological substances which have been used till now can be divided according to the kind of used labels into radioimmunoassay (RIA) using radioactive isotopes as labels, and non-radioactive immunoassays, including enzyme-linked immunosorbent assay (ELISA) using enzymes or fluorescent substances as labels, and fluorescence enzyme immunoassay (FEIA).
The radioimmunoassay among such immunoassays is widely used which shows high sensitivity and is carried out in a precise, simple, easy and rapid manner. Also it has another advantage in that instruments, such as gamma and beta counters, which can be used in this method, are not so expensive.
However, the greatest shortcoming of the immunoassay using radioactive isotopes is that radioactive wastes are released at large amounts. Also this immunoassay is disadvantageous in that the amount of use and the kind of radioactive isotopes are limited by regulations.
In order to solve such problems, enzyme-linked immunosorbent assay (ELISA) is used which is divided into direct ELISA and indirect ELISA according to a labeling type.
Direct ELISA is a way of conducting direct labeling to an antibody or an antibody fragment by a crosslinker, and indirect ELISA is a way of binding hapten to an antibody and conducting measurement using a label recognizing this complex.
Examples of the crosslinker which is used in the direct ELISA includes N,N′-orthophenylenedimaleimide, 4-(N-maleimidomethyl)cyclohexane-N-succinimide ester, 6-maleimidohexane-N-succinimide ester, 4,4-dithiopyridine and the like. Examples of hapten which is used in the indirect ELISA include biotin, dinitrophenyl pyridoxal, fluoresamine and the like, in which biotin uses avidin or streptoavidin as a recognition ligand.
Horseradish peroxidase is mainly used as an enzyme in ELISA, because it can react with many substrates and can be easily bound to an antibody.
In order to verify a labeled enzyme, horseradish peroxidase uses hydrogen peroxide (H2O2) as a substrate solution, and 2,2′-azino-di-[3-ethylbenzothiazoline sulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine, or 3,3′,5,5′-tetramethylbenzidine as a color developer. Also, alkali phosphatase employs orthonitrophenyl phosphate or paranitrophenyl phosphate as a substrate, and β-D-galactosidase uses fluorescein-di-(β-D-galactopyranoside) or 4-methylumbellifery as a substrate.
Meanwhile, if a substance immobilized to a plate is an antibody, ELISA is also called a sandwich ELISA.